The invention relates generally to molecular detection and characterization and more particularly to fabricating reproducible, single-molecule nanopores having controlled geometric properties.
Devices and methods for detecting the passage of a single macromolecule through a nanopore are known. For example, U.S. Pat. No. 5,795,782 to Church et al. describes a nanopore that is created by the insertion of a bacterial pore-forming protein (xcex1-hemolysin) in a lipid membrane. Since protein geometry and physiochemical characteristics are genetically determined, the pore-forming protein is able to form nanopores having a predetermined geometry.
One concern with the prior art techniques is that the process of verifying the existence and proper formation of each nanopore fabricated by using bacterial pore-forming proteins is slow and potentially expensive. Another concern is that because the lipid membrane in which the nanopores are made degrades with time, the resulting nanopores cannot be mass produced for use over an extended period of time.
What is needed is a method for efficiently and consistently forming nanopores with controlled geometries such that the resulting nanopores can be formed in controlled arrays and are capable of being stored for extended periods of time without degrading. What is further needed is such a method that enables precise positioning of the nanopores.
Integrated circuit (IC) fabrication techniques are employed to form precisely dimensioned and positioned nanopores. Film-thickness control within a few atomic monolayers can be achieved by a variety of IC fabrication techniques, including photolithography, epitaxial growth and plasma enhanced chemical vapor deposition (PECVD).
A first embodiment of a method of forming nanopores uses a pair of three-layer segments. Each such multi-layer segment includes a substrate layer, such as a silicon substrate, and includes an intermediate layer and a top layer that are grown or deposited to predetermined and uniform thicknesses. The chemical compositions of the three layers are selected such that the substrate layer and top layer are selectively etchable with respect to the intermediate layer, while the intermediate layer is selectively etchable with respect to the substrate and top layers. In alternate embodiments, each layer is selectively etchable with respect to the other layers. Furthermore, it is contemplated that more than three layers are used to form the segments, with one or more of the layers being selectively etchable with respect the other layers.
The top layer of each three-layer segment is patterned so that at least one supply conduit extends through the top layer to the intermediate layer. Next, the intermediate layer of each segment is patterned using a wet etch process to remove a portion of the intermediate layer. This wet etchant is introduced through the supply conduit and the process is allowed to proceed sufficiently long to form an xe2x80x9cundercutxe2x80x9d in the intermediate layer, with the undercut extending beyond the boundaries of the supply conduit through the top layer.
Following the selective patterning of the top layer and the intermediate layer, excess matter is removed from the edges of each three-layer segment, so that at least one edge of the segment is smooth and is located at a controlled distance from the supply conduit. The smoothed edge is then masked with a photoresist, while leaving a controlled width of the intermediate layer exposed. A narrow slot in the intermediate layer along the smoothed edge of the segment is created using conventional photolithography and etching techniques. After etching, a second selective etch process is conducted to form a path in the intermediate layer from the undercut to the slot. Thus, the completed path within each segment extends from the supply conduit through the intermediate layer to the smooth edge of the segment.
To create a single nanopore, the slots of the pair of three-layer segments are abutted, with the axes through the slots being coaxial while the corresponding layers of the two segments are in non-parallel relationships. The segments are wafer bonded using known techniques. Finally, the exposed portions of the slots are filled. Thus, the resulting structure contains a single nanopore at the interface of the two segments. The geometry of the nanopore is controlled by the orientation of the two segments and the thicknesses of the intermediate layers.
Using modern microchip manufacturing techniques (e.g., epitaxial growth, PECVD, thermal growth, sputtering, evaporation or molecular beam epitaxy (MBE)), the thickness of the intermediate layer can be controlled to the nearest nanometer. Therefore, the dimensions of the resulting nanopore can also be controlled to the nearest nanometer. Furthermore, because the method of manufacture of the individual nanopores involves error-tolerant steps (i.e., process steps that achieve desired results despite process imperfections), both high batch yield and mass production are possible.
In the second embodiment, each of two multi-layer segments is etched to create a recess (or xe2x80x9ctubxe2x80x9d) in its substrate layer. Preferably, the etching process is conducted such that the walls of the recesses intersect the front surface of the substrate layer at steep angles. The recesses are then etched so that the walls are smooth. A second selectively etchable material is blanket deposited or grown to a controlled thickness on each front surface and on the sides and bottom of each recess, thereby forming a coated tub. Each coated tub is then filled with a substance, such as the substrate layer material or a third etchable material. The upper surfaces of the segments are polished to a uniform level, typically past the original surface on which the second etchable material was blanket deposited or grown. As one example, the polishing of the segments may be performed using chemical mechanical polishing (CMP) to remove the materials (e.g., the second and third etchable materials) from the front surfaces of the segments. This step is error-tolerant, since polishing into the front surface of a segment does not adversely affect the process, providing the step leaves a xe2x80x9cfilled tub.xe2x80x9d
For each multi-layer segment, a portion of the second selectively etchable material on the upwardly extending side walls of the filled tub is masked and the exposed portion is at least partially etched. The surrounding substrate material may also be etched to facilitate etching of the second selectively etchable material. The resulting void is filled with a bonding material to bond the substrate to the xe2x80x9cblockxe2x80x9d of material at the center of the recess. The mask is removed and the surface is again polished to a uniform level using polishing techniques known in the IC manufacturing art.
The two multi-layer segments having the same or different nanopore-defining patterns are then aligned such that the second selectively etchable layer of the first segment intersects the second selectively etchable layer of the second segment. The intersection satisfies predetermined geometric criteria. Specifically, the thicknesses of the walls of the tubs determine the area of the nanopore and the shape of the nanopore is determined by the wall thicknesses and by the orientations of the segments. Once aligned, the two segments are wafer bonded using techniques known in the IC manufacturing art.
The back of each multi-layer segment is etched to create a supply conduit. Each supply conduit is etched in a controlled manner from the back side of the segment in a position such that the supply conduit intersects the second selectively etchable layer of the segment. The second selectively etchable layer is then etched out of each segment, thereby creating a nanopore with predetermined dimensions and geometry. xe2x80x9cNanoporexe2x80x9d is defined herein as including pores that have a cross sectional dimension as large as 0.1 millimeter.
An advantage of the invention is that nanopore capability is achieved using techniques that are conventionally considered to be inadequate for such purposes. Since film thicknesses are a key to setting the dimensions of the nanopores and since techniques for controlling film thicknesses to within a few atomic monolayers are known, xe2x80x9ccoarserxe2x80x9d integrated circuit techniques (e.g., photolithography) may be employed for other steps without a sacrifice in end results. Another advantage is that the surfaces of the various channels can be easily modified to optimize their properties for the intended applications. For example, an oxide layer can be created in the channels by performing a baking step within an oxygen-rich environment or by performing anodic oxidation in order to adjust the surface charge for compatibility with DNA. The oxide layer can be further modified using well-known silalyation agents to add chemical functionality and to vary the degree of hydrophobicity. Moreover, the oxide layer can be modified to include affinity probes, such as Biotin and antibodies, enzymes, and/or surface-bound polymers. By tailoring the oxide layer to include agents and/or probes, the invention may be used in chemical analysis and characterization of macromolecules, synthetic and naturally occurring, colloidal micro and nanoparticles, based on interactions of such molecules and particles with the nanopore.